Glucaric acid (tetrahydroxyadipic acid) is a compound discovered long ago in plants and mammals.
In a recent report on high value-added chemicals to be made from biomass (Non-patent Reference 1), the U.S. National Renewable Energy Laboratory listed glucaric acid among the top 12 compounds. This report gives glucaro-γ-lactone, glucaro-δ-lactone, glucarodilactone, and other such lactones (which can be expected to be used as solvents), polyhydroxypolyamides (which can be expected to be used as novel nylons), and the like as examples of glucaric acid derivatives that can be prepared using glucaric acid as a raw material. This report also states that nitric acid oxidation of starch and catalytic oxidation in the presence of basic bleach can be utilized as known methods of producing glucaric acid.
Patent Reference 1 also more recently disclosed transformants capable of biosynthesizing glucaric acid. Specifically, in Patent Reference 1, an Escherichia coli host was transfected by three genes encoding myo-inositol-1-phosphate synthase (Ino1), myo-inositol oxygenase (MIOX), and uronic acid dehydrogenase (udh). It states that the transformants obtained in this way produced glucaric acid in a concentration of 0.72-1.13 g/L in the medium. However, the inventors of Patent Reference 1 held that introduction of an inositol monophosphatase (suhB) gene into the transformants of this patent reference was unnecessary.
Specifically, five activities are theoretically required in a glucaric acid biosynthetic pathway using glucose as a substrate:
activity 1: activity to produce glucose-6-phosphate from a suitable carbon source;
activity 2: activity to convert glucose-6-phosphate into myo-inositol-1-phosphate, that is, inositol-1-phosphate synthase activity;
activity 3: activity to convert myo-inositol-1-phosphate into myo-inositol, that is, phosphatase activity taking myo-inositol-1-phosphate as a substrate
activity 4: activity to convert myo-inositol into glucuronic acid, that is, myo-inositol oxygenase activity; and
activity 5: activity to convert glucuronic acid into glucaric acid, that is, uronic acid dehydrogenase activity. However, since the glucose-6-phosphate that is a product of activity 1 is in fact a metabolic intermediate universally produced by prokaryotic microorganisms, it is not essential to impart this activity to prokaryotic microorganisms.
With regard to activity 3 as well, many microbial strains are known to express endogenous inositol monophosphatase or to have general monophosphatase activity capable of using myo-inositol-1-phosphate as a substrate. It is therefore understandable that no inositol monophosphatase gene was introduced into the transformants of Patent Reference 1.
Therefore, Patent Reference 1 concludes that an inositol monophosphatase gene need not be introduced into transformants to biosynthesize glucaric acid based on metabolic analysis of the transformants produced. Specifically, Patent Reference 1 states “It should also be noted that we did not overexpress the suhB gene or a homologous phosphatase. However, no myo-inositol-1-phosphate was detected among the culture products, while myo-inositol did accumulate. Therefore, we conclude that the phosphatase activity is not limiting flux through the pathway.” (page 33, lines 2-5).
Therefore, there existed no obvious motivation for introducing an inositol monophosphatase gene into transformants for the biosynthesis of glucaric acid.